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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140043	2/3	Homo sapiens	Serum	ExoQuick Filtration	Vargas A	2014	50%
Study summary Full title Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia All authors Vargas A, Zhou S, Éthier-Chiasson M, Flipo D, Lafond J, Gilbert C, Barbeau B Journal FASEB J Abstract Exosomes are extracellular vesicles that mediate intercellular communication and are involved in sev (show more...)Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Filtration Protein markers EV: TSG101/ AChE/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method Filtration steps 0.45µm > x > 0.22µm, Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140043	3/3	Homo sapiens	NAY	(d)(U)C ExoQuick Filtration UF	Vargas A	2014	50%
Study summary Full title Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia All authors Vargas A, Zhou S, Éthier-Chiasson M, Flipo D, Lafond J, Gilbert C, Barbeau B Journal FASEB J Abstract Exosomes are extracellular vesicles that mediate intercellular communication and are involved in sev (show more...)Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration UF Protein markers EV: TSG101/ AChE/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140043	1/3	Homo sapiens	NAY	(d)(U)C Filtration	Vargas A	2014	44%
Study summary Full title Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia All authors Vargas A, Zhou S, Éthier-Chiasson M, Flipo D, Lafond J, Gilbert C, Barbeau B Journal FASEB J Abstract Exosomes are extracellular vesicles that mediate intercellular communication and are involved in sev (show more...)Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ AChE/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Wash: volume per pellet (ml) 10 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140043
species	Homo sapiens
sample type	Serum	Cell culture	Cell culture
cell type	NA	NAY	NAY
medium		EV Depleted	EV Depleted
condition	NAY	NAY	NAY
separation protocol	ExoQuick Filtration	(d)(U)C ExoQuick Filtration UF	(d)(U)C Filtration
Exp. nr.	2	3	1
EV-METRIC %	50	50	44